The monitoring of pharmacokinetics, genetic, cellular, molecular or other types of events in vivo is of great interest to monitor drug or gene therapy efficacy as well as disease status or progression in small laboratory mammals and in the human body. In this respect, fluorescence imaging, both in vitro and in vivo, has been used extensively to generate anatomical and functional information from within cells and organisms.
Fluorescence imaging of internal parts of animals (including humans) for anatomical or functional purposes often involves the injection of an extrinsic fluorophore, typically chemically coupled with another molecule, that distributes within the animal and accumulates preferentially in cells and organs of interest. Images are then acquired by detecting the fluorescence and mapping the signal relative to the anatomy of the animal. However, the excitation and emission spectra of such extrinsic fluorophores often overlap with those of intrinsic fluorophores such that the fluorescence signal is a combination of the signals from each fluorophore. Furthermore, such studies are often conducted using more than one extrinsic fluorophores which may have overlapping spectra. As a result, fluorescence images often contain undesirable signals that obscure the signal from the fluorophore of interest.
Methods commonly used to attenuate or eliminate unwanted fluorescence signals are based on spectral differences of the fluorescence emission, fluorescence lifetime differences (e.g. FLIM), or frequency domain hardware techniques. All of them have limitations. Methods based on spectral difference are limited to fluorophores having emission spectra that do not significantly overlap thereby allowing acquisition of fluorescence at a non-overlapping wavelength which is specific for a particular fluorophore. Methods based on fluorescence lifetime help distinguish signals from different fluorophores but do not retain the information related to fluorophore intensity and consequently information related to concentration of the fluorophore is lost. Frequency domain hardware techniques require multiple image acquisition at a plurality of phase delays to suppress unwanted fluorescence and are therefore time consuming.
Accordingly, it would be desirable to be provided with a fluorescence imaging method overcoming the above-mentioned deficiencies.